aprABC: a Mycobacterium tuberculosis complex-specific locus that modulates pH-driven adaptation to the macrophage phagosome

Following phagocytosis by macrophages, Mycobacterium tuberculosis (Mtb) senses the intracellular environment and remodels its gene expression for growth in the phagosome. We have identified an acid and phagosome regulated (aprABC) locus that is unique to the Mtb complex and whose gene expression is induced during growth in acidic environments in vitro and in macrophages. Using the aprA promoter, we generated a strain that exhibits high levels of inducible fluorescence in response to growth in acidic medium in vitro and in macrophages. aprABC expression is dependent on the two-component regulator phoPR, linking phoPR signalling to pH sensing. Deletion of the aprABC locus causes defects in gene expression that impact aggregation, intracellular growth, and the relative levels of storage and cell wall lipids. We propose a model where phoPR senses the acidic pH of the phagosome and induces aprABC expression to fine-tune processes unique for intracellular adaptation of Mtb complex bacteria.     

Sulfolipid-1 biosynthesis restricts Mycobacterium tuberculosis growth in human macrophages

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.     

Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D''Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.     

The PhoP-PhoQ two-component regulatory system of Photorhabdus luminescens is essential for virulence in insects

Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg(2+)-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg(2+)-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.     

The mmaA2 gene of Mycobacterium tuberculosis encodes the distal cyclopropane synthase of the alpha-mycolic acid

Infection with Mycobacterium tuberculosis (Mtb) remains a severe global health problem that has prompted an aggressive search for new antibiotic targets and vaccine strategies for this persistent pathogen. Recently, a wide variety of genetic determinants of Mtb pathogenicity have been identified, including several genes involved in the biogenesis of the complex Mtb cell envelope. Among these are the mycolic acid cyclopropane synthases, a family of proteins that modify the major cell envelope lipids of Mtb with a diversity of cyclopropane rings. Despite substantial sequence identity, these proteins catalyze highly specific cyclopropane modifications, including proximal modification of the alpha-mycolate (pcaA) and trans-cyclopropane modification (cmaA2). Here we report the mycolic acid modification function of a third cyclopropane synthase, mmaA2, through the creation and analysis of an M. tuberculosis mmaA2 null mutant. Unexpectedly, mmaA2 is essential for the distal cyclopropane modification of the alpha-mycolate, a function previously attributed to cmaA1. alpha-Mycolates of a cmaA1 null mutant were unaffected, demonstrating that cmaA1 is not required for alpha-mycolate modification. Although fully cyclopropanated methoxymycolates are produced in the mmaA2 mutant, cis-cyclopropanation is impaired, leading to accumulation of unsaturated methoxymycolate derivatives. This study establishes mmaA2 as a distal cyclopropane synthase of the alpha-mycolates of M. tuberculosis and the first cyclopropane synthase to modify both alpha- and oxygenated mycolates. These results expand our knowledge of the biosynthesis of the Mtb cell envelope and will allow further elucidation of the relationship between Mtb pathogenesis and the fine structure of mycolic acids.     

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.     

Mycobacterium tuberculosis uses host triacylglycerol to accumulate lipid droplets and acquires a dormancy-like phenotype in lipid-loaded macrophages

Two billion people are latently infected with Mycobacterium tuberculosis (Mtb). Mtb-infected macrophages are likely to be sequestered inside the hypoxic environments of the granuloma and differentiate into lipid-loaded macrophages that contain triacylglycerol (TAG)-filled lipid droplets which may provide a fatty acid-rich host environment for Mtb. We report here that human peripheral blood monocyte-derived macrophages and THP-1 derived macrophages incubated under hypoxia accumulate Oil Red O-staining lipid droplets containing TAG. Inside such hypoxic, lipid-loaded macrophages, nearly half the Mtb population developed phenotypic tolerance to isoniazid, lost acid-fast staining and accumulated intracellular lipid droplets. Dual-isotope labeling of macrophage TAG revealed that Mtb inside the lipid-loaded macrophages imports fatty acids derived from host TAG and incorporates them intact into Mtb TAG. The fatty acid composition of host and Mtb TAG were nearly identical suggesting that Mtb utilizes host TAG to accumulate intracellular TAG. Utilization of host TAG by Mtb for lipid droplet synthesis was confirmed when fluorescent fatty acid-labeled host TAG was utilized to accumulate fluorescent lipid droplets inside the pathogen. Deletion of the Mtb triacylglycerol synthase 1 (tgs1) gene resulted in a drastic decrease but not a complete loss in both radiolabeled and fluorescent TAG accumulation by Mtb suggesting that the TAG that accumulates within Mtb is generated mainly by the incorporation of fatty acids released from host TAG. We show direct evidence for the utilization of the fatty acids from host TAG for lipid metabolism inside Mtb. Taqman real-time PCR measurements revealed that the mycobacterial genes dosR, hspX, icl1, tgs1 and lipY were up-regulated in Mtb within hypoxic lipid loaded macrophages along with other Mtb genes known to be associated with dormancy and lipid metabolism.     

Pharmacological and genetic activation of cAMP synthesis disrupts cholesterol utilization in Mycobacterium tuberculosis

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3’, 5’-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies.